Although immunologic tolerance is controlled in part through thymic deletion, mechanisms of peripheral tolerance must be in place to avoid autoreactive aggression of T cells that survive into the periphery. One proposed mechanism of peripheral tolerance is the process of clonal anergy, a hyporesponsive state resulting from engagement of the TCR in the absence of costimualtory signals. Although anergy has been studied for many years, the precise molecular mechanism to explain the functional defects in anergic T cells remains unclear. We previously demonstrated defective TCR-induced Ras/MAP kinase signaling in anergic T cells. Using a novel adenoviral transduction system, we recently have shown that constitutively active (CA) Ras restores IL-2 production and MAP kinase activation in anergized T cells, arguing that defective Ras activation is causally linked to hyporesponsiveness. To understand the mechanism for blunted Ras activity in anergic cells we performed a gene array screen that has identified several attractive candidates. One of these is diacylglycerol kinase (DGK). However, whether upregulated DGK is sufficient to explain all the features of anergy is not clear, and the role of other potential candidates is not known. In Specific Aim 1, whether upregulation of DGK is sufficient to explain all features of the anergic state will be investigated. These experiments will be facilitated by the use of Coxsackie and adenovirus receptor (CAR) transgenic T cells and adenoviral vectors encoding wildtype and kinase-dead DGK isoforms. In Specific Aim 2, other attractive gene candidates will be considered, focusing on Schlafen and CRTAM. Adenoviral constructs will be made to study whether normal T cell activation will be suppressed. Monoclonal antibodies will be generated against CRTAM to study regulation of protein expression and effects of ligation or blockade on T cell function. New candidates will also be identified using EST arrays. Ultimately, a complete understanding of the anergic state on the molecular level should guide the development of novel pharmacologic therapies to promote or reverse peripheral tolerance in vivo. [unreadable] [unreadable]